1

A dual reporter system for in situ detection of plasmid transfer in aerobic and 1

anaerobic conditions 2

Jaroslaw E. Król, Linda M. Rogers, Stephen M. Kronea, Eva M. Top

* 3

4

Department of Biological Sciences, Initiative for Bioinformatics and Evolutionary Studies, 5

University of Idaho, PO Box 443051, Moscow, ID 83844-3051 6

a Department of Mathematics, Initiative for Bioinformatics and Evolutionary Studies, University 7

of Idaho, Moscow, ID 83844-1103 8

9

10

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Running title: Plasmid transfer detection by a dual reporter system. 12

13

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*Corresponding author, E-mail: evatop@uidaho.edu, Phone: 1- 208-885-5015; Fax: 1-208-885-15

7905. 16

Copyright © 2010, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Appl. Environ. Microbiol. doi:10.1128/AEM.00226-10 AEM Accepts, published online ahead of print on 7 May 2010

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Abstract 1

We designed a new genetic tool to detect plasmid transfer in anaerobic and aerobic conditions. 2

The system is based on the T7RNA polymerase gene and a pT7-driven oxygen-independent 3

green fluorescent protein, evoglow®, alone or in combination with red fluorescent protein 4

DsRed. Constructs are available as plasmids and mini-mariner transposons. 5

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Introduction 1

Conjugative plasmid transfer allows exchange of genetic material within and between 2

bacterial populations through direct cell-to-cell contact and plays an important role in bacterial 3

adaptation. This is exemplified by the rapid spread of multi-drug resistance (8) and the 4

abundance of diverse transferable pollutant degradation plasmids (14). While many 5

environmental factors are known to affect plasmid transfer efficiency (5), few studies have 6

addressed the effect of oxygen levels on plasmid transferability, in spite of growing concerns 7

about antibiotic resistance and virulence among facultatively and strictly anaerobic pathogens (2, 8

3, 10, 12, 13). There is thus an obvious need for new methods that allow quantitative analysis of 9

plasmid transfer under both aerobic and anaerobic conditions. 10

The traditional method of plasmid transfer detection is based on plating and enumeration 11

of donor, recipient and transconjugant cells on selective media. However, this approach does not 12

allow in situ observation of conjugation in spatially structured populations like bacterial 13

microcolonies and biofilms. Therefore it cannot address questions about the effects of spatial cell 14

organization on plasmid spread. In order to detect plasmid transfer in situ confocal laser scanning 15

microscopy (CLSM) is often used to detect either fluorescently labeled molecular probes or 16

fluorescent proteins encoded by chromosome- or plasmid-located reporter genes (11). Here the 17

challenge is the level of fluorescence, which may depend on many factors. For the frequently 18

used GFP family of fluorescent proteins, an important limiting factor is the concentration of 19

oxygen necessary for protein maturation (9). These fluorescent proteins are therefore not useful 20

in anaerobic or low-oxygen conditions, such as sediments, anaerobic digesters, the human gut, 21

and thick biofilms. 22

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Recently, a new flavin mononucleotide (FMN)-based oxygen-independent fluorescent 1

protein, called evoglow® (EcFbFP), has been described (4). We propose application of this 2

fluorescent protein to provide a true representation of plasmid transfer in situ that is not 3

confounded by the effects of O2 concentration on fluorescence levels. 4

To allow in situ monitoring of plasmid transfer in both aerobic and anaerobic 5

environments, we constructed a dual reporter system comprised of the fluorescent reporter 6

proteins DsRed and EcFbFP. The red fluorescent protein DsRed (also referred to as RFP) 7

provides very strong and stable fluorescence but requires oxygen in the maturation process (1). It 8

can thus serve as an internal control to confirm conditions of limited oxygen. The system, 9

presented in Fig. 1 consists of two independent elements: the phage T7 RNA polymerase gene 10

and the other is a reporter gene (or genes) driven by the T7 promoter (pT7). Thus when T7 RNA 11

polymerase is not present, the cells show no fluorescence at all. However, when both the 12

polymerase and the reporter genes are present in the same cell, the latter are expressed and 13

fluorescence is detected. Both components are available on plasmid vectors or as a part of the 14

mariner mini-transposon (Supplemental Fig. S1). 15

The T7 RNA polymerase gene was amplified from plasmid pBT20-∆bla-T7pol (6) and 16

cloned into pGem3Zf+ under two promoters, the lactose operon promoter plac and pT7, 17

(resulting in plasmids pGemT7pol, pGemT7cat in Fig. S1a), and subsequently into the mariner 18

mini-transposon delivery vector pBT20-∆bla (6), resulting in pMaT7cat plasmid (Fig. S1b). The 19

presence of two strong promoters should ensure expression of the T7 RNA polymerase gene 20

under most growth conditions. Detailed descriptions of cloning strategy and methods used in this 21

paper are described in Supplemental Materials. 22

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The second part of the system consists of a broad-host-range plasmid or mariner 1

transposon with one or two reporter genes driven by the pT7 promoter. Plasmid pGLOW-Txn-2

Bs2 (Fig. S1c), which contains the EcFbFp gene driven by pT7 promoter, was obtained from 3

evocatal GmbH (Düsseldorf, Germany). Vector pEvoGlowRed encodes both EcFbFP and DsRed 4

expressed using a single pT7 promoter (Fig. S1d). This construct can be mobilized into a broad 5

range of strains that can be used as recipients in plasmid transfer studies. The vector can also be 6

used as a promoter probe vector, since part of the pGem3Zf+ cloning site (MCS) is located 7

between pT7 and the dsRed-EcFbFP genes. Single PstI, SalI, XbaI and BamHI sites can thus be 8

used to clone foreign DNA fragments and detect promoter activity under both aerobic and 9

anaerobic conditions in bacterial strains not containing the T7 RNA polymerase gene. 10

Since the reporter plasmids described above are based on the broad-host-range vector 11

pBBR1MCS (7), which can replicate and persist in many Gram-negative bacteria, problems due 12

to occasional plasmid loss and mobilization could be encountered. To avoid this, and to further 13

extend the range of organisms in which our system can be used, the gene cassettes containing 14

either EcFbFP or both dsRed and EcFbFP driven by the pT7 promoter were also cloned into the 15

mariner transposon delivery vector pBT20-∆bla (6), resulting in constructs carrying different 16

antibiotic resistance genes (Fig. S1e-k). The mariner transposon can be used for in vivo as well 17

as in vitro mutagenesis, and has been successfully applied in Gram-negative and Gram-positive 18

bacteria (15). 19

To quantify the efficiency of plasmid transfer in relation to the parental strains, one 20

typically also counts donor and/or recipient cells. To simultaneously detect recipients and 21

transconjugants in aerobic conditions with one vector, we first constructed the vector pGlowRed. 22

In this vector the plac-dsRed cassette was cloned into the EcoRV site of the pGLOW-Txn-Bs2 23

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vector (Fig. S1e). In this case, expression of the dsRed gene is constitutive, driven by the lac 1

promoter as well as the distantly located pKm promoter (red fluorescence), but EcFbFP is 2

expressed only in T7 polymerase-carrying strains (data not shown). Thus, when pGlowRed is 3

present in the recipient, these cells show only red fluorescence, but transconjugants are green and 4

red (yellow). In parallel a mariner transposon containing a constitutively expressed dsRed gene 5

and pT7EcFbFP with Gm and Km resistance genes was constructed (data not shown). 6

To evaluate the feasibility of using EcFbFP and DsRed in plasmid transfer studies in both 7

aerobic and anaerobic conditions, we monitored the transfer of plasmid F’, as it is known to 8

transfer efficiently in both aerobic and anaerobic conditions (12). The plasmid was first marked 9

using pMaT7cat. In two marked plasmids (F’::T7cat1, F’::T7cat2) insertions were located within 10

the lacI gene of the F’ plasmid (See Materials and Methods in Supplemental Materials). Thus, 11

the locations of the MaT7cat transposon in this region do not disturb any of the core genes 12

involved in replication, maintenance/control or transfer functions. Transfer of the marked 13

plasmids to three E. coli K12 recipient strains was observed in simple cross-streak experiments 14

(Fig. S2). The first two recipient strains carried reporter plasmids pGLOW-Txn-Bs2 and 15

pEvoGlowRed; the third strain carried a chromosomal insertion of the mini-transposon 16

MaGlowRedKm (E. coli K12-GR). Donor and recipient precultures were grown anaerobically, 17

then cross-streaked on LB plates and incubated in aerobic or anaerobic conditions at 37ºC. After 18

aerobic incubation transconjugants were observed as bright green fluorescent cells when 19

recipients carried pGLOW-Txn-Bs2, and as both green and red fluorescent cells when recipients 20

contained pEvoGlowRed (Fig. 2a, b). The same results were obtained with aerobically grown 21

precultures (data not shown). In anaerobic conditions only green fluorescence was observed for 22

both types of recipients (Fig. 2c, d). Similarly, green and red fluorescence in the presence, and 23

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only green fluorescence in the absence, of oxygen was detected when E. coli K12-GR was used 1

as the recipient (data not shown). The lack of red fluorescence under anaerobic conditions was 2

expected and served as an internal control to confirm conditions of limited oxygen. Green color 3

intensity and surface areas of fluorescent transconjugants were qualitatively similar in aerobic 4

and anaerobic conditions. Both marked F’ plasmids showed identical results. Additional filter 5

matings followed by plate counting confirmed that there was no obvious effect of the absence of 6

oxygen on F’ plasmid transfer under these experimental conditions. The average frequency of 7

transconjugants/recipients was not significantly different between anaerobic and aerobic 8

conditions (0.5 and 0.8; p<0.05, Standard t-test). 9

We also validated the use of evoglow® in thick cell layers by growing a colony of E. coli 10

constitutively expressing EcFbFP and RFP on agar plates in alternating aerobic/anaerobic 11

conditions (see Supplemental Methods). The cross section micrograph shows that EcFbFP was 12

expressed all the way down to the agar while the RFP signal was detected only at the edge of the 13

colony (Fig. S3). 14

One caveat of our system is the requirement for recipient strains that contain either the 15

reporter gene(s) or the T7 RNA polymerase gene, thus limiting its use in experiments aimed at 16

monitoring plasmid transfer into indigenous bacterial populations. However, one can detect 17

plasmid transfer from an exogenous donor if indigenous bacteria are first isolated, marked with a 18

reporter or MaT7pol system, and then reintroduced into their ecosystem. 19

In conclusion, we present here the first fluorescent reporter system that allows in situ 20

monitoring of plasmid population dynamics under both aerobic and anaerobic conditions with 21

simultaneous verification of low-oxygen conditions through lack of red fluorescence. So far, all 22

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other reporter systems used to monitor plasmid transfer were based on GFP and RFP and thus 1

depend on the presence of oxygen. 2

The research was supported by NIH Grant 1R01 GM73821; the Olympus FV1000 3

Multiphotone Confocal Microscope was funded by the M.J. Murdock Charitable Trust (grant # 4

2006-127JVZ022207) and an NIH Small Instrumentation Grant (1S10RR02260601). We thank 5

Dr. L. Forney for helpful suggestions, Dr. A. Paszczynski and K. H. Kucharzyk for use of their 6

anaerobic chamber, and L. Sherick and K. Turner for technical assistance. We are also grateful to 7

Dr. J.-M. Ghigo for providing plasmid F’, and to Prof. T. T. Hoang for pBT20-∆bla-T7pol and 8

pBT20-∆bla. 9

JEK designed the dual reporter system, constructed all vectors, performed the plasmid 10

transfer experiments and wrote the manuscript draft. EMT, LMR, and SMK oversaw the project, 11

provided help with the experimental design and data interpretation, and assisted in writing the 12

manuscript. 13

References. 14

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oligomerization of DsRed, a red fluorescent protein from coral. Proc. Natl. Acad. Sci. U.S.A. 16

97:11984-11989. 17

2. Biebricher, C. K., and E. M. Duker. 1984. F and type 1 piliation of Escherichia coli. J. 18

Gen. Microbiol. 130:951-957. 19

3. Curtiss, R. I., L. G. Caro, D. P. Allison, and D. R. Stallions. 1969. Early Stages of 20

Conjugation in Escherichia coli. J. Bacteriol. 100:1091-1104. 21

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4. Drepper, T., T. Eggert, F. Circolone, A. Heck, U. Krausz, J.-K. Guterl, M. Wendorff, A. 1

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13. Strohmaier, H., R. Noiges, S. Kotschan, G. Sawers, G. Högenauer, E. L. Zechner, and 1

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1

Fig. 1. Schematic action of the reporter system. Donor strain contains plasmid F’ marked with 2

the T7 RNA polymerase gene, and recipient strains contain the vector pGLOW-TXN-Bs2 (left) 3

or pEvoglowRed (right). Transconjugant cells with both the marked F’ and a reporter gene are 4

green fluorescent (left) or green and red (yellow; right) respectively. 5

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Fig. 2. Microscopy images of mating mixtures containing either E. coli MG1655Rif (F’::T7cat) 7

(donor) and MG1655Nal (pGLOW-TXN-Bs2) (recipient 1, a and c), or the same donor and 8

MG1655Nal (pEvoGlowRed) (recipient 2, b and d). Mixtures were incubated for 48 hours under 9

aerobic (a, b) and 72 hours under anaerobic (c, d) conditions. Left panel: evoglow®, green 10

fluorescence; right panel: DsRed, red fluorescence; middle panel: overlay of both channels. 11 at Univ of Idaho on June 4, 2010

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